Abstract: Mutations in phosphatidylinositol (PI) phosphatases cause diseases including cancer and neurodegenerative disorders. A common assumption is that failure to dephosphorylate a specific lipid substrate underlies these diseases. Emerging evidence raises the possibility that PI phosphatases have lipid- phosphatase-independent functions that contribute to disease pathology when these enzymes are mutated. In support of this hypothesis, mutations that inactivate some PI phosphatases result in phenotypes that are different from a total loss of the protein. This proposal will test the hypothesis that conformational changes that accompany the regulation of the activity of PI phosphatases influence their interactions with protein partners. The disease related PI phosphatases PTEN and Fig4 will be used as model enzymes. The mentored phase of this proposal will take place in the laboratory of Dr. Lois Weisman, a leading expert in the phosphatidylinositol 3,5-bisphosphate (PI3,5P2) signaling pathway. In pursuit of the proposed research, the applicant will be trained in large scale genetic screens in yeast, genetic incorporation of unnatural amino acids for probing dynamic protein-protein interactions (with Dr. Ann Mapp, U. Michigan), and mammalian tissue culture. In addition to research, the mentored phase will include: writing scientific papers, presentations at national and/or international meetings, mentoring of undergraduate and graduate rotation students, and participation in local journal and research clubs. In the independent phase, the applicant will establish an academic laboratory where the applicant proposes to use both yeast and mammalian systems to elucidate principles of cross-talk between cellular pathways via lipid-phosphatase-independent functions of PI phosphatases. Aim 1 will characterize unpublished Fig4 mutants that either activate or inhibit Fab1 kinase activity and screen for new mutants. In Aim 2 these mutants will be used to determine how Fig4 regulates PI3,5P2 via regulation of the Fab1/Vac14/Fig4 complex. Fig4 domains involved in protein-protein interactions will be probed through incorporation of photo-activatable amino acid cross-linkers in Fig4 using nonsense suppression. The potential for Fig4 to act as a protein phosphatase will be tested by identifying phosphorylation sites altered in cells expressing wild-type versus catalytically inactive Fig4. These and future studies will test the hypothesis that specific stimuli, which induce conformational changes in yeast Fig4, in turn activate or inhibit Fab1. Aim 3 will determine the roles of the PTEN active site in a regulatory intramolecular interaction with the C-terminal tail. Binding assays between PTEN active site mutants and variants of the C-terminal tail will be performed both in intact cells and with recombinant proteins. In addition, proteins that specifically interact with active or inactive PTEN conformations will be identified via purification of native complexes associated with TAP-tagged mutants from intact cells. These studies will test the hypothesis that conformational changes regulating PTEN lipid phosphatase activity serve in parallel to regulate downstream pathways through protein-protein interactions.